ABSTRACT (PUBLICLY RELEASABLE) The current level of research activity involving gene therapy with either synthetic vectors (carriers) or engineered viruses is unprecedented. Liposomes are the most widely studied nonviral carriers worldwide for nucleic acid (NA) and drug delivery applications. Cationic liposomes (CLs) are relatively safe nonviral vectors used in ongoing clinical trials. CLs may either be complexed via electrostatic interactions with therapeutic NAs (anionic DNA or short interfering RNA) for gene delivery and silencing, or used as vectors of potent cytotoxic hydrophobic drugs, encapsulated within their lipid bilayer, in cancer therapeutics. Among the biggest advantages of nonviral vectors (over viral vectors which are currently more efficient in in vivo settings) are their safety, their low immunogenicity and their ability to transfer entire genes (containing coding and noncoding sequences) and regulatory sequences into cells (currently not feasible with engineered viruses because of capsid size limitations). The development of nonviral lipid-based vectors with efficacy competitive with viral vectors in vivo will require a mechanistic understanding of how synthetic vectors may be functionalized to overcome the major intracellular hurdle of endosomal escape. Successful endosomal escape is required for release of therapeutic nucleic acid within the cell cytosol and therefore maximum efficacy. The first aim of our current award is to employ modern biophysical and synthetic approaches to the rational design of functionalized CL?NA nanoparticles (NPs) with synergistic, complementary dual-function PEG-lipid and fusogenic components for optimized endosomal escape. Modern methods of organic and solid phase chemistry will be employed to synthesize dual-function PEG-lipids with cell targeting and endosome escaping properties. The second aim of our current award is to optimize efficacy of a new class of CL-based carriers of the hydrophobic drug paclitaxel (PTX) for cancer therapeutics. (PTX is among the most widely used cancer chemotherapy drugs to treat ovarian, breast, lung, pancreatic, and other cancers and is included in the World Health Organization?s List of Essential Medicines.) This will be achieved by developing a mechanistic understanding of the relation between physical and chemical properties of the carrier (i.e. size of the functionalized CL carrier, membrane spontaneous curvature, and lipid tail structure) and functional efficacy (i.e. PTX membrane solubility, cell uptake of vector and PTX delivery leading to cytotoxicity against human cancer cells). The broad, long-term objective of our research is to develop a fundamental science base through mechanistic studies that will lead to the design and synthesis of nonviral vectors of nucleic acids and hydrophobic drugs for gene and cancer therapeutics. We use fluorescence live-cell imaging with quantitative particle tracking to visualize complex pathways and interactions with cells, and established assays in low and high serum conditions to measure transfection efficiencies (for CL-NA vectors) and NP antitumor efficacy (for CL-hydrophobic drug based vectors). Synchrotron X-ray diffraction (and when required cryo-TEM) is used for structure determination. Our capabilities in custom synthesis via modern methods of organic chemistry are essential for the success of both goals. Successful outcomes in our fundamental mechanistic studies (major aims 1 and 2) will have a major impact on the creation of efficient and safe CL vectors of nucleic acids and hydrophobic drugs with anti-tumor activity.